逆转录酶抑制剂AZT对胶质瘤干细胞端粒酶活性和细胞增殖的影响

Effects of AZT on the proliferation of human brain glioma stem cell and the related mechanisms

目的探讨逆转录酶抑制剂3-叠氮-3-脱氧胸腺核苷(AZT)对脑胶质瘤干细胞增殖的影响及相关机制。方法原代分离培养脑胶质瘤干细胞和脑胶质瘤细胞并鉴定,两种细胞同时设立为实验组(0.125 mol/L、0.250 mol/L、0.500 mol/L AZT)和对照组。MTT法检测AZT对两种细胞增殖的影响;流式细胞仪检测细胞凋亡和细胞周期的改变;端粒重复序列扩增技术-酶联免疫吸附试验(TRAP-ELISA)检测端粒酶活性的变化。结果 AZT对两组细胞的生长抑制呈浓度-时间依赖性(均P<0.05),在同一浓度和时间点,AZT对脑胶质瘤干细胞的生长抑制作用弱于脑胶质瘤细胞(均P<0.05)。0.250 mol/L、0.500 mol/L AZT作用72 h后,脑胶质瘤干细胞的凋亡率分别为(4.21±1.53)%、(10.60±0.38)%,而脑胶质瘤细胞的凋亡率分别为(6.75±1.25)%、(14.30±2.59)%,明显高于胶质瘤干细胞(均P<0.05)。AZT对两组细胞端粒酶活性的抑制呈浓度-时间依赖性(均P<0.05),且在同一浓度和时间点对脑胶质瘤细胞的作用明显强于脑胶质瘤干细胞(均P<0.05)。结论逆转录酶抑制剂AZT对脑胶质瘤干细胞和脑胶质瘤细胞均有明显的生长抑制作用,可能机制是通过抑制端粒酶活性、调控细胞周期和诱导细胞凋亡实现。脑胶质瘤干细胞的耐药性强于胶质瘤细胞,其机制有待进一步研究。

Objective To investigate the effect of reverse transcriptase inhibitor,3-azido-3-deoxythmidine（AZT）,on the proliferation of human glioma stem cells and the related mechanisms.Methods Primary cultured brain glioma stem cells and glioma cells were isolated,cultured and identified.The glioma stem cells were divided into experimental groups（0.125,0.250,and 0.500 mol/L AZT） and control group,while the same groups were set up in glioma cells.The effects of AZT on proliferation of two kinds of cell were detected by MTT assay.The cell apoptosis and cell cycle were examined by flow cytometry（FCM）.Telomerase activity was determined by telomeric repeat amplification protocol-enzyme linked immunosorbent assay（TRAP-ELISA）.Results AZT inhibited the growth of two kinds of cells in concentration-and time-dependent way（all P〈0.05）.The growth inhibitory effect of AZT on glioma stem cells was weaker than glioma cells at the same concentration and time point（all P〈0.05）.The apoptosis rates were 4.21±1.53%（0.250 mol/L AZT） and 10.60±0.38%（0.500 mol/L AZT） in glioma stem cells 72 h after the treatment with AZT,while the correspondent rates were 6.75±1.25%（0.250 mol/L AZT） and 14.30±2.59%（0.500 mol/L AZT） in glioma cells which were significantly higher than those in glioma stem cells（all P〈0.05）.AZT inhibited telomerase activity of two kinds of cell in concentration-and time-dependent way（all P〈0.05）.The inhibitory effect of AZT on glioma cells was significantly stronger than on glioma stem cells in the same concentration and time point（all P〈0.05）.Conclusions AZT can inhibit the proliferation of glioma stem cells and glioma cells,which may be related to inhibition of telomerase activity,regulation of cell cycle and induction of cell apoptosis.The drug resistance of glioma stem cells is stronger than glioma cells,and the mechanisms need further study.